PROJECT SUMMARY/ABSTRACT: In this KO1 proposal, we attempt to deeply interrogate the role of molecular recognition and structure in a receptor-ligand system, type I Interferons (IFNs), that has fundamental implications for basic receptor signaling biology as well as human health. We intend to elucidate the structure- functional relationships of IFN induced signaling and to enhance IFN functions through combinatorial, computational, and structural biology. The training and research proposal describes a five year mentored program under the guidance of Dr. K. Christopher Garcia designed to build upon Dr. Mendoza's previous training in order to further expertise in combinatorial, computational, and structural biology. This will establish Dr. Mendoza as an independent multi-disciplinary scientist with a powerful set of tools to give him the experimental range to tackle problems in cytokine engineering for cancer biology. We will attempt to understand how IFN cytokines mediate a wide range of physiological processes through their effects on cell growth, differentiation, and proliferation. Type I IFNs have anti-proliferative properties important for the surveillance and control of malignant cells. Upon engagement of their receptor extracellular regions, IFNs differentially activate intracellular JAK/STAT signaling cascades. Members of the type I IFN family elicit different signaling responses despite binding to a common pair of receptors, IFNR1 and IFNR2. Fundamentally, the specificity of cytokine signaling is largely determined by the composition of receptor chains within the signaling complex. While much is known about cytokine-activated intracellular signaling pathways, we know much less about how extracellular structural changes induced by ligand binding translates into signaling events. The experiments proposed will provide more clarity to this question. The potential translational and cancer therapeutic applications are based on a pioneering study published more than ten years ago suggesting that IFN diversification through in vitro evolution could yield molecules with enhanced functional properties. Now, armed with powerful new methodologies, we wish to revisit this concept to create IFNs with diverse functional properties, but also to provide a structural and mechanistic rationale for these altered activities in a way that could inform strategies to engineer IFNs for cancer therapy in the future. We will achieve this by 1) in vitro evolution of IFN1, 2) identification of variants with altered function using in vitro anti-viral and anti-proliferative assays, 3) structural characterization of the ternary complexes of the evolved IFNs to correlate structure with function, and 4) the computational design and in vitro evolution of chimeric cytokines with distinct and novel signaling and functional outputs. By understanding the molecular basis of cytokine/receptor interactions with correlated function, the results from this work may be able to guide the future development of cytokines as therapeutics as well as expand the capabilities and diversity of receptor mediated signaling, functional activities, and therapeutic properties.